Immunoreactive peptide sequence from a 43 KD human cancer antigen

ABSTRACT

A tumor associated protein has been discovered to exhibit immunoreactivity to human monoclonal antibody L92. A ten amino acid peptide segment of the protein and a fourteen amino acid peptide which contains the ten amino acid peptide has also been isolated and exhibits immunoreactivity to the same antibody. Using truncated fusion proteins, the minimum recognition site for antibody binding was determined to be four amino acids. Also disclosed are polypeptide compositions against human tumors that includes the polypeptides or protein of the present disclosure, as well as antibodies reactive with these polypeptides that may be employed directly for treatment or diagnosis. A certain embodiment of the present invention is also the DNA sequence encoding the ten amino acid peptide and the DNA encoding the fourteen amino acid peptide which also exhibits immunoreactivity to human monoclonal antibody L92 and cytotoxic T cells.

The government owns rights in the present invention pursuant to grantnumbers CA12582, CA56059, and CA30647 from the National Institutes ofHealth.

This application is a continuation-in-part of application Ser. No.08/115,170, filed Aug. 31, 1993 now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of immunotherapybased on the identification and isolation of human tumor antigens. Inparticular embodiments, the invention also relates to the use of humanmonoclonal antibodies to isolate the epitopic sequence of atumor-associated antigen. Further embodiments relate to syntheticpeptides derived from tumor-associated proteins and their uses fortherapy, diagnosis and prognosis of human cancer.

2. Description of the Related Art

The ideal tumor vaccine is one that induces anti-tumor immunity withoutadverse effects. Ideally, such a vaccine would be stable, inexpensive toproduce and easily administered. Early attempts with whole cell vaccinesor cellular extracts, have improved the survival of cancer patientssignificantly. However, problems still exist with the production,storage and delivery of the vaccines. For example, there still existsthe possibility of contamination by cellular molecules that may induceside effects in patients.

Because of these problems, synthetic or recombinant antigens arecontemplated to be more effective vaccines, provided the antigendelivery system, carrier molecules, and adjuvants are optimized for theinduction of specific anti-tumor immunity. What is needed is theidentification and characterization of tumor antigens that playimportant roles in tumor destruction in vivo, in particular thosecapable of inducing T-cell immunity in humans.

Because elevated titers of autoantibodies are known to be present inhosts with various autoimmune diseases, neuropathological diseases andcancers, they have been used to screen cDNA clones from expressionlibraries in order to discover pathogenic antigens of such diseases (TanE. M., 1991; Amagai M. et al., 1991; Dropcho E. J. et al., 1987; SzaboA. et al., 1991; Hayashibe K. et al., 1991). The difficulties of usingthis approach to identify human tumor associated antigens (TAA) includethe unavailability of high titer human anti-TAA antibodies and theinterference of serum antibodies unrelated to TAA.

Melanoma associated antigens (AU and FD) unique to autologous melanomacells have been detected using autologous sera (Carey T. E. et al.,1979). The FD antigen is a 90 kD cell-surface glycoprotein. Theantigenic determinant is present on an ion-binding protein with aminoacid homology to transferrin (Real F. X. et al., 1984). The samemolecule was identified by murine monoclonal antibodies developedagainst the human melanoma-associated antigen p97 (Brown J. P. et al.,1982). Although the sequence of p97 is known, the sequence of the uniqueFD epitope has not yet been determined. The Hellstrom laboratory hasinvestigated the 97,000 MW glycoprotein antigen, p97, that ispredominantly expressed by human melanoma cells (Woodbury R. G. et al.,1980; Brown J. P. et al., 1981). The p97 gene has been inserted into arecombinant vaccinia virus and has induced strong anti-tumor immunityagainst mouse melanoma cells transfected to express the p97 antigen(Brown J. P. et al., 1982). A clinical trial with this vaccine isongoing by this group (Hellstrom I. et al., 1992).

Vlock and colleagues used autologous melanoma sera after dissociatingserum immune complexes via acidic treatment and found an antigenic 66 kDacidic glycoprotein (Vlock D. R. et al., 1988). Subsequent epitopeanalysis has shown that the carbohydrate moiety of the glycoproteinrepresents its antigenic determinant. The sequence of the core proteinhas not been reported.

Allogeneic polyclonal sera from melanoma patients have also been used toidentify immunogenic melanoma associated antigens. These antigens areshared by more than one melanoma. Bystryn and colleagues detectedimmunogenic melanoma associated antigens (200+, 150, 110, 75, and 38 kD)using sera from patients who received immunotherapy with melanoma cellsupernatant (Li J. et al., 1990).

Gupta and associates have defined a urinary tumor associated antigen(UTAA), which is a glycoprotein antigen originally found in the urine ofmelanoma patients and also found on the melanoma cell surface, includingthe M14 cell line. The UTAA is comprised of several subunits that arelinked together by disulfide bonds. The total molecular weight isapproximately 300 kD, with immunogenic subunits of 45 kD, 65 kD, 90 kD,120 kD and 150 kD (Euhus D. M. et al., 1990).

Ferrone and colleagues used pooled sera from melanoma patients toidentify a 50 kD glycoprotein antigen (D-1) in melanoma (Hayashibe K. etal., 1991). Using cDNA libraries of a melanoma cell line, theimmunoreactive clone was isolated and sequenced. The successful cloningwas possible after extensive absorption of non-specific antibodies withE. coli proteins and blocking of cDNA plaques on nitrocellulose with IgGisolated from the pooled sera of healthy donors. While the exact tumorspecificity of this protein (D-1) is not known, northern blothybridization has shown that peripheral blood lymphocytes (PBL) andnormal fibroblasts do not express this antigen.

Several other studies have identified protein molecules from other humancancer cells that migrate at or around the 43 kD position in SDS-PAGEWestern blot analysis. These studies have used murine or humanmonoclonal antibodies. HGP43 is a human glycoprotein of 43 kDa, whichwas identified by a murine monoclonal antibody and was originallyreported as a protective antibody against lethal Listeria monocytogenesinfection in mice (Fontan E. et al., 1992). HGP43 is detected in theurine of healthy, normal individuals as well as in cancer patients.HGP43 has also been shown to stimulate mouse monocytes to inducecytotoxicity against the Lewis lung tumor (Fontan E. et al., 1993). Theamino acid sequence from HGP43 that is reactive to the monoclonalantibody has not been reported.

The human monoclonal antibodies 16.88 and C-OU 1 react with another 43kD protein molecule in human cancer cells (Erb K. et al., 1991). Theantigen is most strongly expressed in melanoma and less strongly incolon cancer cells. The amino acid sequence of this protein is partiallyknown and exhibits about 70% identity with cytokeratin 18.

Another HuMAb, MS2B6, has been used to detect a cytoplasmic antigenpresent at high density in ovarian carcinoma and is less prevalent in avariety of human cancers and in certain types of normal tissues (SmithL. H. et al., 1992). On SDS-PAGE Western blot analysis, MS2B6 reacted toproteins with a broad range of molecular weights, 33-44 kD and 60 kD.Competitive inhibition studies have shown no crossreactivity between theantigen identified with MS2B6 and the cytokeratin identified with HuMAbs16.88 and C-OU 1.

Another approach to developing anti-tumor vaccines is the development ofanti-idiotype vaccines. In this method, MAbs are developed against anantigen of interest. These antibodies are then used to screen an epitopelibrary to find a peptide that mimics the target antigen. This anti-idepitope is then used as a vaccine. This is particularly important indeveloping antibodies to glycosidic antigens which cannot be easilysynthesized. Dr. Ferrone's laboratory has pursued an investigation ofanti-id vaccines mimicking high molecular weight melanoma associatedantigens (HMW-MAAs) for the active specific immunotherapy of melanoma(Kusama M. et al., 1989). The HMW-MAAs are expressed in high density bymelanoma cells, but have a restricted distribution on normal tissues(Reisfeld R. A. and Cheresh D. A., 1987). Anti-ids mimicking HMW-MAAswere developed using syngeneic murine monoclonal antibodies to MAAs asimmunogens. When these anti-id vaccines were injected in mice andrabbits, specific immune responses were demonstrated (Kusama M. et al.,1989; Challopadhyay P. et al., 1991). Injection of these vaccines intopatients during a Phase I clinical trial has shown that the anti-idvaccine is safe and has induced clinical responses in patients withmelanoma including 2 complete remissions (Mittelman A. et al., 1992).However, the high anti-tumor antibody or T-cell responses observed inanimal models have not been replicated in clinical trials. The weakimmune responses seen in patients by immunization with these antigensmay reflect, therefore, the fact that these antigens may be partiallytolerated under normal immunologic conditions in humans.

Therefore, there still exists an immediate need for an anti-tumorimmunotherapy. An effective vaccine would comprise tumor antigens thatcan induce antibody and/or T cell immune responses in humans.

SUMMARY OF THE INVENTION

The present invention seeks to overcome these and other drawbacksinherent in the prior art by identifying and isolating protein antigensthat are highly immunogenic in humans. These antigens are then used invaccines to promote specific anti-tumor responses.

An important embodiment of the present invention is a purifiedpolypeptide which has been found to be immunoreactive with HuMAb L92.More particularly, this embodiment of the invention is a purifiedpolypeptide that includes the 4 amino acid segment KYQI (seq id no:8),or the purified polypeptide that includes the 9 amino acid segmentQDLTMKYQI (seq id no:9) or even a purified polypeptide that includes the10 amino acid segment QDLTMKYQIF (seq id no:1). The purified polypeptideof the invention may be further defined as including the sequencesdelineated as seq id nos:1, 8 and 9 and comprising a molecular mass ofabout 43 kDa as defined by the mobility of the protein through adenaturing polyacrylamide gel relative to proteins of known molecularmass.

The term "purified polypeptide" as used herein, is intended to refer toa polypeptide composition, isolatable from other tumor cell associatedproteins, wherein the polypeptide is purified to any degree relative toits naturally-obtainable state, i.e., in this case, relative to itspurity within a tumor cell extract. A purified polypeptide thereforealso refers to a polypeptide, free from the environment in which it maynaturally occur in intact cells.

In certain embodiments, "purified" will refer to a polypeptidecomposition which has been subjected to fractionation to remove variousnon-protein components such as other cell components. Various techniquessuitable for use in protein purification will be well known to those ofskill in the art. These include, for example, precipitation withammonium sulphate, PEG, antibodies and the like or by heat denaturation,followed by centrifugation; chromatography steps such as ion exchange,gel filtration, reverse phase, hydroxylapatite and affinitychromatography; isoelectric focusing; gel electrophoresis; andcombinations of such and other techniques.

In certain other embodiments, the purified polypeptide will bechemically synthesized by solid phase synthesis and purified away fromthe other products of the chemical reactions by HPLC for example.Alternatively, the polypeptide may be produced by the overexpression ofa DNA sequence included in a vector in a recombinant cell. In thismethod of producing the polypeptide, purification would be accomplishedby any appropriate technique mentioned in the preceding paragraph.

It is understood that other peptide segments of the 43 kDa protein mayalso be immunoreactive with HuMAb L92. For example, it is known thatantigenic determinants are sometimes discontinuous and therefore anotherpeptide segment of the same 43 kDa protein, may also be immunoreactivewith the HuMAb L92. All such peptide segments either continuous ordiscontinuous, and even including the full length protein which isrecognized by HuMAb L92 are understood to be encompassed by the presentinvention and to lie within the scope of the present claims. Forexample, a purified polypeptide which includes the sequences designatedherein as seq id nos:1, 8 or 9 and having a length of less than 100amino acids, or having a length of less than 50 amino acids, or having alength of less than 25 amino acids or even defined as having a length of10 amino acids or less would also be included within the scope of thepresent claimed invention.

It is also understood that a protein or polypeptide segment of thepresent invention may also be fused, generally by genetic techniqueswell known to those of skill in the art, to a carrier protein and thatsuch a protein-protein or protein-peptide fusion which exhibitsimmunoreactivity with HuMAb L92 is also encompassed by the presentinvention. For example, a polypeptide that contains any of the aminoacid sequences designated herein as seq id nos:1, 8 or 9 and furtherincluding the amino acid sequences of β-galactosidase orglutathione-S-transferase, for example would also be an embodiment ofthe present invention. It is understood that these carrier proteinsequences are mentioned by way of example only and that other knowncarrier protein sequences such as keyhole limpet hemocyanin (KLH),bovine serum albumin (BSA), other albumins such as ovalbumin, mouseserum albumin or rabbit serum albumin or any other suitable proteinsequence may also be included as embodiments of the present invention.It is further understood that the use of the term "protein" does notlimit the invention to polypeptides or peptides of any particular size.Peptides from as small as several amino acids in length to proteins ofany size as well as protein-peptide fusions are encompassed by thepresent invention, so long as the protein or peptide is antigenic and/orexhibits low immunogenicity.

In a general sense, the proteins or polypeptides of the presentinvention may be defined as being associated with, or derived from humantumor cells. Even more particularly, the proteins and peptides of thepresent invention may be defined as a protein of M_(r) of about 43 kDaand associated with human tumor cells which is immunoreactive with HuMAbL92; any polypeptide fragments of said 43 kDa protein; protein fusionsor peptide-protein fusions comprising any part of the said 43 kDaprotein; and even more particularly, a protein-peptide fusion capable ofimmunoreactivity with HuMab K92 in which a polypeptide, preferably oneof those peptide sequences designated as seq id nos:1, 8 or 9 is fusedwith β-galactosidase or GST. It is understood that using the techniquesof recombinant DNA, any portion of the sequence of the 43 kDa proteinincluding the entire coding sequence and any promoters, enhancers, polyA splice site, ribosome binding site or other transcription ortranslation control sequences may be inserted into a foreign DNAsequence and expressed in order to obtain a protein withimmunoreactivity to HuMAb L92 and that any such use of the geneticand/or amino acid sequences of the 43 kDa protein are encompassed by thepresent claimed invention.

It is further understood that the amino acid sequence or underlyinggenetic sequence of the protein of the present disclosure may be alteredby for example, site directed mutagenesis, or by any other means and aprotein may be obtained thereby which retains its biological utility andantigenicity. It is also understood that such altered proteins may notretain the biological activity, or they may possess altered or enhancedbiological function or antigenicity and that all such altered proteinsare encompassed by the present invention.

In a further embodiment, the present invention relates to a recombinantcell line capable of producing the proteins, polypeptides and/orprotein-protein or protein-polypeptide fusions discussed in thepreceding paragraphs. The said cell line may preferably be a bacterialcell line, such as an E. coli cell line which contains within the cellsa plasmid or viral vector comprising a DNA segment encoding the protein,peptide or fusion of the present invention and the control elementsnecessary for the replication of the vector and for expression of thepolypeptide, protein or fusion. It is understood that the cell line mayalso be another bacterial cell, or a yeast, plant, animal or even ahuman cell line. The selection of the appropriate cell line and acompatible vector are well known to those of skill in the art and allsuch cell lines and vectors fall within the scope of the presentinvention. It is also understood that the expression of the geneencoding the protein may be under the control of an inducible promoter,for example the lac promoter and that expression may be controlled byexogenously applied inducers. Most preferably, the peptides designatedherein as seq id nos:1, 8 and 9 are fused to β-galactosidase, under thecontrol of the lac promoter and expressed in an E. coli cell line.

As used herein, the term "recombinant" cell line is intended to refer toa cell into which a recombinant gene, such as a gene encoding apolypeptide immunoreactive with human MAb L92 has been introduced.Therefore, engineered cells are distinguishable from naturally occurringcells which do not contain a recombinantly introduced gene. Engineeredcells are thus cells having a gene or genes introduced through the handof man. Recombinantly introduced genes will either be in the form of acDNA gene (i.e., they will not contain introns), a copy of a genomicgene, or will include genes positioned adjacent to a promoter notnaturally associated with the particular introduced gene.

Transformed cells are generally understood to be those cells which havean inserted plasmid vector which is capable of replication within thesaid transformed cells. Transfected cells are generally understood to bethose which have been infected with a viral vector or a virally derivedvector. In both cases, the vector may carry a segment of DNA whichencodes for the protein or peptide of interest and which is capable ofbeing replicated and expressed along with the DNA of the plasmid orviral vector. It is sometimes possible through manipulation of thegrowth conditions of the cells to "overproduce" the desired protein orpeptide such that it is the major protein expressed in the cell.

Generally speaking, it may be more convenient to employ as therecombinant gene a cDNA version of the gene. It is believed that the useof a cDNA version will provide advantages in that the size of the genewill generally be much smaller and more readily employed to transfectthe targeted cell than will a genomic gene, which will typically be upto an order of magnitude larger than the cDNA gene. However, theinventor does not exclude the possibility of employing a genomic versionof a particular gene where desired.

Another embodiment of the present invention is a segment of DNA whichencodes the polypeptide sequence designated herein as seq id no:1, 8 or9 and its complement, which DNA sequences are depicted in FIG. 4 anddesignated seq id nos:6 and 7. It is understood that these DNA sequencesmay be used as probes to identify complementary stretches of DNA from anorganism or from a vector and that the identified DNA segment may beisolated and cloned by techniques well known in the art, and that allsuch use of the said DNA segments and the products isolated with the useof said DNA segments are encompassed by the present invention. It isalso understood that minor changes in the said DNA sequences may occurthrough genetic code redundancies, for example, and that such geneticsequences, which encode the said peptide sequences are also included asa part of the claimed invention.

Another embodiment of the present invention is a human antibody and morepreferably a monoclonal antibody immunoreactive with the polypeptidesequences designated herein as seq id nos:1, 8 or 9. The peripheralblood lymphocytes (PBL) of a melanoma patient were used to establishB-cell lines that each produce an antibody to a tumor associated antigenwith Epstein-Barr Virus (EBV) transformation techniques (Irie et al.,1982). One of these cell lines produces human monoclonal antibody L92.The said antibody, (HuMAb) L92, was shown to react to a 43 kD proteinassociated with human tumor cells. This 43 kD protein appears to be anew and unique tumor associated antigen in that it is distinguished frommost of the known melanoma associated antigens discussed above by virtueof its relative molecular weight (M_(r)) of 43 kDa, and the availablesequence data.

For example, the 10 amino acid sequence reactive to HuMAb L92 did notmatch the sequence of any reported protein including cytokeratin 18.This distinguishes HuMAb L92 from the human monoclonal antibodies 16.88and C-OU 1. These later two antibodies react with another 43 kD proteinmolecule in human cancer cells whose amino acid sequence is partiallyknown and which exhibits about 70% identity with cytokeratin 18. BecauseHuMAb L92 exhibits no identity with the cytokeratin 18, it is believedthat these two antibodies recognize different molecules or epitopes thandoes HuMAb L92.

The similarity of HuMAb L92 to MS2B6 is not known, but theimmunoreactive band with HuMAb L92 on Western blotting has produced asharp and clear band of 43 kD, indicating that HuMAb L92 detects adifferent antigenic epitope than does MS2B6. Since the amino acidsequences of these 43 kDa or near 43 kDa proteins are not known, exactcomparison is not possible. The possibility that these antigens detectdifferent epitopes on the same molecule has not been excluded. This willbe clarified when a full length amino acid sequence of the protein(s) isdetermined.

To identify the gene encoding the antigenic epitope, a cDNA expressionlibrary constructed from the human melanoma cell line UCLASO M14 (Irieet al., 1976) was screened with HuMAb L92. DNA sequence analysis of theisolated clone revealed that the antigenic epitope was a 10 amino acidpeptide. Further testing revealed that a four amino acid peptide(Lys-Tyr-Gln-Ile, seq id no:8) contained within the 10 amino acidpeptide is the minimum antigenic epitope of HuMAb L92. The peptide wasexpressed in E. coli as a β-galactosidase-peptide fusion, and as aglutathione-S-transferase (GST)-protein fusion.

A cDNA expression library is a group of vectors containing cloned DNAsegments, cloned generally from poly A⁺ mRNA, such that only activelytranscribed message is cloned into the library. Thus, the DNA segmentsinserted in the library vectors are complementary only to RNAs that areactively transcribed or expressed in the particular cell line from whichthe library is derived.

The human monoclonal antibody referred to herein as HuMAb L92 is anotherembodiment of the present invention. The antibody is expressed by aB-cell line developed by the inventor and is immunoreactive with the 43kDa melanoma associated antigen and peptides designated herein as seq idnos:1, 2 and 8. It is understood that this antibody is useful forscreening samples from human patients for the purpose of detectingmelanoma associated antigens present in said samples. The said antibodymay also be useful in the screening of expressed DNA segments orpeptides and proteins for the discovery of related antigenic sequences.All such uses of the said antibody and any antigens or epitopicsequences so discovered fall within the scope of the present invention.

Another embodiment of the present invention is an antigen compositioncomprising a protein or polypeptide which is immunoreactive with HuMAbL92 in an amount effective to elicit a CTL or antibody response.Preferably the polypeptide composition will include the polypeptidesequences designated herein as seq id nos:1, 8 or 9, singly or incombination and may also include the sequence of a carrier protein suchas β-galactosidase. It is contemplated that the polypeptide will bepresent in the composition at a concentration of between 1 mg/ml andabout 10 mg/ml and preferably at about 5 mg/ml. The polypeptidecomposition discussed in the present paragraph may also be a componentof a polyvalent melanoma cell vaccine (MCV).

A polyvalent melanoma cell vaccine is for example, a vaccine whichcomprises several melanoma cell lines which express multiple melanomaassociated antigens. The cells are rendered inviable, preferably byirradiation, and administered to a patient in order to elicit an immuneresponse as discussed elsewhere (Morton et al., 1992, incorporatedherein by reference).

As is shown herein, the melanoma cells of the MCV may be rendered moreeffective by pre-immunization with the peptide segment designated seq idno:1. Therefore, the cells may or may not be pretreated, and thepolypeptide composition which includes the polypeptide sequences of thepresent invention may be administered in conjunction with the MCV. It iscontemplated that a method of treating human cancer patients andpreferably, human melanoma patients would comprise administering to saidpatients a polyvalent melanoma cell vaccine about every two weeks forthree times and then about once a month for about a year, followed byadministration about every 3 months for about four times and then aboutevery six months thereafter, and further comprising administering acomposition comprising the polypeptide sequence herein designated as seqid no:1 or no:8 or no:9 about every four weeks for two to four times andthen about every six months thereafter.

Alternatively, the present invention may be a method of enhancing theimmune response in a subject comprising the steps of obtaining cytotoxiclymphocytes from said subject, contacting said cytotoxic lymphocyteswith a polypeptide according to seq id no:1, seq id no:8 or seq id no:9and reintroducing said lymphocytes into said subject. The enhancedimmune response may be an active or a passive immune response.Alternatively, the response may be part of an adoptive immunotherapyapproach in which the lymphocytes are pulsed with the polypeptide andthen reintroduced into the subject. In certain embodiments, the subjectis a human cancer patient and more preferably a human melanoma patient.The lymphocytes may be obtained from the serum of the subject, oralternatively from tumor tissue to obtain tumor infiltrating lymphocytesas disclosed in Rosenberg et al., Science, vol 233, page 1318,incorporated herein by reference. In certain preferred embodiments, thelymphocytes are peripheral blood lymphocytes and in other embodiments,the method of enhancing the immune response is practiced in conjunctionwith melanoma whole cell therapy.

It is understood that although clinical applications for the polypeptideand nucleic acid sequences, antibodies and recombinant cell lines of thepresent invention are disclosed, that the various embodiments of theinvention will have other important utilities, such as the screening oftissue and culture samples for the presence of melanoma associatedantigens and in the development of new therapeutic and prophylacticagents for use against melanoma. Further, the polypeptides andantibodies of the present invention are useful for advancing the generalknowledge and use of antigenic sequences, antibodies and activatedcellular immune systems in the search for immunogenic weapons againstvarious diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A, Western blot of M14 cells with HuMAb L92.

FIG. 1B, Western blot of M14 cells with non-specific human IgM.

FIG. 1C, SDS-PAGE analysis of proteins stained with Coomasie BrilliantBlue.

In FIGS. 1A-C: Lane 1, molecular weight standard; Lane 2, M14 Humanmelanoma cell line grown in human AB serum; Lane 3, M14 human melanomacell line grown in media containing fetal calf serum; Lane 4, M12 humanmelanoma cell line grown in media containing fetal calf serum; Lane 5,fetal brain, second trimester.

FIG. 2. Reactivity of HuMAb L92 to ₃₅ S-methionine-labeled M14 humanmelanoma cell line. Biotin-labeled anti-human IgM antibody was used as asecond antibody. Lane 1, whole cell extract; Lane 2, immunoprecipitatewithout HuMAb L92; Lane 3, immunoprecipitate with HuMAb L92; Lane 4,immunoprecipitate with HuMAb L612.

FIG. 3A. Expression of immunopositive fused protein in E. coli analyzedby SDS-PAGE of the protein stained with Coomasie Brilliant Blue.

FIG. 3B. Expression of immunopositive fused protein in E. coli analyzedby Western blot using HuMAb L92.

In FIGS. 3A-B: Lane 1, E. coli lysogen containing immunopositive #810clone-protein, seq id no:2; Lane 2, same as lane 1 without IPTGinduction; Lane 3, β-galactosidase; Lane 4, molecular weight standard.

FIG. 4. The double stranded nucleotide sequence and deduced amino acidsequence of immunopositive clone #810. The amino acid sequence is thesequence designated seq id no:2. The upper DNA sequence is designatedseq id no:6 and the lower, complementary sequence is designated seq idno:7.

FIG. 5A. Competition of synthetic peptide (DSRPQDLTMKYQIF, seq id no:2)for the binding of HuMAb L92 to the protein of the melanoma cell lineanalyzed by immunostaining HuMAb L92 pre-absorbed with the syntheticpeptide, seq id no:2.

FIG. 5B. Competition of synthetic peptide (DSRPQDLTMKYQIF, seq id no:2)for the binding of HuMAb L92 to the protein of the melanoma cell lineanalyzed by immunostaining with HuMAb L92 post-absorbed with thesynthetic peptide, seq id no:2. In FIGS. 5A-B: Lane 1, molecular weightstandard; Lane 2, lysogen from clone #810; and Lane 3, M14 melanomalysates.

FIG. 6. Reactivity of HuMAb L92 to synthetic peptide (QDLTMKYQIF, seq idno:1) in ELISA. A 10 amino acid peptide was immobilized on ELISA plates(Reacti-Bind™, PIERCE), according to the manufacturer's instructions.▪=HuMAb L92: =10 μg/ml of unrelated human IgM (L612); □=without HuMAbL92.

FIG. 7A. Detection of the 43 kD protein on the surface of the M14 cellby Western blot analysis. The L92 HuMAb antibody was incubated with M14cells (233×10¹⁰ cells) overnight at 4° C. After centrifugation, thesupernatant antibody was tested for its reactivity to the 43 kD proteinand #810, seq id no:2 fused protein.

FIG. 7B. Control reaction of detection of the 43 kD protein on thesurface of the M14 cell by Western blot analysis in which L92 HuMAb wasincubated overnight without M14 cells.

In FIGS. 7A-B: Lane 1, molecular weight standard; Lane 2, M14 celllysates; Lane 3, #810 antigen, seq id no:2.

FIG. 8A. Diffuse cytoplasmic signals are visible in melanoma M14 cells(Magnification×200). The cells are hybridized against the #810 antisenseprobe.

FIG. 8B. The melanoma M14 cells are hybridized against the #810 senseprobe.

FIG. 9A. Cytotoxicity against autologous BCL pulsed with #810 peptide,seq id no:1. Cytotoxicity and proliferation assays were performed asdescribed herein. Representative data of 19 patients and 19 controlnormal donors are shown. At 0, 4 and 8w, a patient's PBMC show positivecytolyses (≧17% at E:T of 80:1) of autologous #810-pulsed BCL whilethose of a normal donor do not. Lysis of autologous BCL without peptidesdid not exceed 7% by any effector. Mean cpm±SD of triplicate culturesare shown in (B). At 4 and 8w, values of the patient's PBMC with #810peptide, seq id no:1, 0.001-20μM are significantly higher than thosewithout #810, seq id no:1 (p<0.005) and maximal SIs were 2.71 and 2.87at 4 and 8w, respectively whereas significant proliferative response to#810, seq id no:1, is not observed before vaccination (0w) and in acontrol donor.

FIG. 9B. Proliferative response to #810, seq id no:1 of PBMC from avaccinated melanoma patient. Assays were done as described in 9A.

FIG. 10. Cytotoxicity of a patient's PBMC restimulated with #810, seq idno:1 in vitro. PBMC from patient A 4w after vaccination were in vitrorestimulated with #810 and were assayed for cytolyses of autologous#810, seq id no:1--pulsed BCL and melanoma MA targets as describedherein. □ and ∘ indicate cytotoxicity of control PBMC cultured withmedium only. Lysis of autologous BCL without peptides or pulsed with anirrelevant decapeptide did not exceed 8% at an E:T of 80:1 by an eithereffector. Lysis of K562 by #810, seq id no:1--restimulated PBMC andcontrol at E:T of 80:1 were 24.0 and 24.5%, respectively. Data arerepresentative of three separate studies.

FIG. 11A. Cytolysis of autologous #810, seq id no:1--pulsed BCL. Coldtarget inhibition of cytolyses of autologous #810, seq id no:1--pulsedBCL and melanoma MA. PBMC from patient A were restimulated with #810,seq id no:1 similarly to FIG. 10 and assayed for cytolyses of autologous⁵¹ Cr-labeled #810, seq id no:1--pulsed BCL (A) and melanoma MA (B) atE:T of 20:1. Unlabeled cold target cells were added at indicated ratios.Lysis of BL without peptides was 3.0% at E:T of 20:1. Data arerepresentative of four separate experiments.

FIG. 1B. Cytolysis of melanoma MA as described in FIG. 11A.

FIG. 12. Antibody inhibition of cytotoxicity by #810, seq idno:1--restimulated PBMC. #810, seq id no:1--restimulated PBMC frompatient A were assayed for cytolyses of autologous #810, seq idno:1--pulsed BCL and melanoma MA at E:T of 40:1 in the presence orabsence of antibodies. A final concentration of each antibody was 10μg/ml. Lyses of autologous BCL without peptides and pulsed with anirrelevant decapeptide were 5.4 and 5.2%, respectively. Data arerepresentative of four separate experiments.

FIG. 13. Cytolyses of autologous and allogeneic #810, seq idno:1--pulsed BCL by #810, seq id no:1--restimulated PBMC. Representativedata of three experiments are shown as % specific cytolysis at E:T of40:1 against #810, seq id no:1--pulsed BCL targets. Effectors are #810,seq id no:1--restimulated PBMC of patient B (HLA-A2+) and D (HLA-A11+).Parentheses of left margin indicate HLA-A or B antigens shared betweenthe effectors and targets. Each autologous and allogeneic target wasalso preincubated in media only or pulsed with an irrelevantdecapeptide, and was tested for lysis by the effectors. Lysis of thosecontrol targets did not exceed 5% by an either effector.

FIG. 14. Determination of minimal antibody recognition peptide. SeveralGST-fused peptides were truncated from the 10 amino acid HuMAb L92immunoreactive peptide sequence (seq id no:1) were prepared usingsynthetic oligonucleotides and pGEX-2T as the vector. While thetruncation of 1 amino acid residue from the C-terminus did not affectthe binding of HuMAb L91 (Lane 3), the removal of 2 C-terminal aminoacid residues completely deleted antibody binding (lane 4). The deletionof 6 amino acid residues from the N-terminus of the 10 amino acidpeptide did not affect the binding of HuMAb L92 (Lane 7), but theremoval of 7 N-terminal amino acids again deleted antibody binding (Lane8).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The inventors have previously developed a number of B-cell lines thatproduce human monoclonal antibodies reactive to melanoma cell lines(Irie et al., 1982) using Epstein-Barr virus (EBV) transformationtechniques. One particular B-cell line produced HuMAb L92 from amelanoma patient's peripheral blood lymphocytes (PBL). HuMAb L92 is anIgM-class antibody that reacts to several types of human cancer cellsbut does not respond to normal cells by the immunoadherent assay whichspecifically detects surface antigens (See Table 1). The ability ofantigen(s) to bind with HuMAb L92 was not altered by treatment withglycosidases such as β-galactosidase, α-mannose, neuraminidase, andα-fucosidase. HuMAb L92 was devoid of reactivity to gangliosides andneutral glycolipids purified from antigen-positive melanoma cells. Thisindicated that L92 recognized a protein or peptide antigen.

HuMAb L92 was used to identify and clone a gene encoding theimmunoreactive epitope of a 43 kD protein associated with human cancercells. The epitope-encoding gene encodes a newly discovered 10amino-acid sequence (seq id no:1), as well as a 4 amino acid sequence(seq id no:8) contained within the 10 amino acid segment which has beenexpressed in the form of a protein-peptide fusion with β-galactosidaseand GST in E. coli. HuMAb L92 specifically bound to both theprotein-peptide fusion and the synthetic peptide alone. Absorption ofHuMAb L92 with melanoma cells has proven that the antigen ispredominantly expressed inside the cell of certain melanomas, while ahigh density of the antigen is expressed on the cell surface of othermelanomas.

The nucleotide sequence encoding the peptide was determined by dideoxysequencing, a technique well known to those of skill in the art. Thissequence was screened for homology to all known sequences contained inthe Genebank database and no homology was found between the clonedsequence and any other reported DNA sequence.

M14 lysates were prepared after the cells were pulsed with ³⁵S-methionine for 4 hrs. After the cell lysate was pre-cleaned withanti-human IgM-biotin and Streptavidine agarose, HuMAb L92 was added andincubated. Either the unrelated HuMab L612 (Yamamoto S. et al., 1990),or buffer alone, was added to the precleaned cell lysate. The immunecomplex was then precipitated with anti-human IgM-biotin andStreptavidine agarose, and the precipitates were subjected to SDS-PAGEfollowed by autoradiography. Immunoprecipitates with HuMAb L92 showed aclear band at 43 kD. However, the immunoprecipitate, either with HuMAbL612 or without primary antibody, failed to show the band (FIG. 2),indicating that the 43 kD protein band in lane 3 specifically binds toHuMAb L92.

Western blot analysis showed that the protein-peptide fusion boundspecifically to HuMAb L92. An antigen-encoding peptide of 10 amino acidswas synthesized and tested for its immunoreactivity in vitro. HuMAb L92reacted specifically to the 10 amino acid peptide in both an antibodyinhibition assay and a solid-phase ELISA. The HuMAb L92 also was shownby Western blot analysis to react with the four amino acid peptidedesignated as seq id no:8. These results suggest that the identifiedpeptide sequences comprise immunogenic epitopes of the 43 kD proteinthat induces immune responses in man.

Abbreviations

The following abbreviations are used throughout the present disclosure.HuMAb, human monoclonal antibody; ELISA, enzyme-linked immunosorbentassay; PBL, peripheral blood lymphocytes; FCS, fetal calf serum; PCR,polymerase chain reaction; IPTG, isopropyl thiogalactosidase; SDS-PAGE,sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TAA,tumor-associated-antigens; CTL, cytotoxic T lymphocyte; MCV, melanomacell vaccine; MAA, melanoma-associated antigen; BCL, Epstein-Barrvirus-transformed B lymphoblastoid cell line; IA, immunoadherence; PBMC,peripheral blood mononuclear cells; ³ H! TdR, tritiated thymidine; SI,stimulation index; LAK, lymphokine-activated killer.

Synthetic peptides

Peptide #810 (QDLTMKYQIF, seq id no:1) and an non-immunogenicdecapeptide of irrelevant sequence (IMTQLFQDYK, seq id no:5) weresynthesized in the Beckman Research Institute of the City of Hope(Duarte, Calif.) using the 9'-fluorenylmethoxycarbonyl (FMOC) method.Those peptides were purified by high-pressure liquid chromatography. Thepeptides were more than 95% pure. The identity of the peptides wasconfirmed by a high resolution mass spectrum.

Tumor cell lines

Melanoma cell lines, gastric, colon, lung and breast cancer cell lines,erythroleukemia K562 and Epstein-Barr virus transformed B lymphoblastoidcell lines (BCL) were all cultured by RPMI 1640 medium (JRH Biosciences,Lenexa, Kans.) supplemented with 10% heat-inactivated fetal bovine serum(Gemeni Bioproducts, Calabasas, Calif.).

Western blotting

Cell lysate was analyzed for the presence of 43 kD protein using L92antibody by Western blotting. Briefly, cells were harvested, pelletedand dissolved in lysis buffer (50 mM Tris-HCl, 5% β-mercaptoethanol, 2%sodium dodecyl sulfate, 0.1% bromophenol blue, 10% glycerol). Proteinsof cell lysates were separated by polyacrylamide gel electrophoresis inreducing condition. Proteins in a gel were transferred to anitrocellulose filter. Filters were incubated in phosphate bufferedsaline containing 5% bovine serum albumin, washed and incubated withprimary antibody L92, washed and then incubated withperoxidase-conjugated goat anti-human IgM secondary antibody (BoehringerMannheim), washed and then coloring reaction was performed by4-chloro-1-naphtol in methanol with H₂ O₂.

Immunoadherence (IA) assays

IA assays were performed as described herein. Briefly, target cells wereincubated with antibody L92 for 90 min at 37° C., washed and thenincubated with a guinea pig complement (Whittaker M. A. Bioproducts,Inc., Walkersville, Md.) for 10 min. After complete settling of targetcells and erythrocytes for 20 min, erythrocyte rosetting around targetcells were examined.

Allogeneic whole melanoma cell vaccines (MCV)

Melanoma patients may be immunized with MCV as previously reported(Morton et al., 1992). MCV consists of for example, three melanoma celllines (M10, M24 and M101), which are irradiated and cryopreserved beforeuse. The MCV is thawed, washed and mixed with BCG and then injectedintradermally every 2 weeks ×3, then monthly for 1 year, and then every3 months ×4, finally every six months.

Peripheral blood mononuclear cells (PBMC) from melanoma patients andnormal donors

In order to obtain PBMC, patients were bled before (0w) and at monthlyintervals (4, 8 weeks) after the initiation of vaccination. PBMC wereseparated by Ficoll-Hypaque gradient centrifugation and cryopreservedbefore testing. PBMC from normal donors were obtained from American RedCross (Los Angeles, Calif.). BCL were prepared by transformation ofthose PBMC with Epstein-Barr virus.

Culture medium

RPMI 1640 supplemented with 10% heat-inactivated human AB serum (IrvineScientific, Santa Aria, Calif.) was used as culture medium in thefollowing examples.

Monoclonal antibodies

Human IgM monoclonal antibodies L92 and L612 (anti-ganglioside GM₃) werecloned and purified in the inventor's laboratory. Murine IgG monoclonalantibodies anti-HLA class I, anti-HLA-DR, anti-CD3, anti-CD4 andanti-CD8 were purchased from AMAC, Inc., Westbrook, Me. ⁵¹ Cr labeledtargets were preincubated with anti-class I, anti-HLA-DR, L92 or L612for 1 h at 37° C. before the addition of effector cells. Effector cellswere preincubated with anti-CD3, CD4 or CD8 for 1 h before the additionof targets. Those antibodies were used at the final concentration of10μg/ml in 6 h-cytotoxicity assays. The results were evaluated forstatistical significance by the Student's t test.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

EXAMPLE I Immunochemical Analysis

Two methods of immunochemical analysis were performed in order toidentify antigen molecules which were reactive with the HuMAb L92. Theseincluded separation of protein solutions by SDS-polyacrylamideelectrophoresis followed by Western Blot analysis, in which the proteinsare transferred to a nitrocellulose filter and reacted to an antibodywhich is conjugated to another antibody capable of a colorimetricreaction. The antigens were also subjected to ELISA reactions in whichthe peptide was placed in microtiter plate wells and reacted with theantibody which was then reacted with the secondary, indicator antibody.

SDS-Polyacrylamide Western Blot Analysis

Human melanoma cell lines M14 and M12, and human fetal brain cells weretested for their reactivity to HuMAb L92 in Western blotting. Twodifferent media supplements, FCS and human AB serum, were used in thecase of the M14 melanoma cell line. To prepare the lysates, cells werelysed in a lysis solution containing NP-40, SDS and deoxycholate.

SDS-polyacrylamide gel electrophoresis followed by Western blot analysiswas carried out to detect antigen molecules reactive to HuMAb L92.Sample protein solutions in 50 mM Tris-HCl (pH 6.8; 5%β-mercaptoethanol, 2% SDS, 0.1% BPB, 10% glycerol) were boiled for 5min. Protein separation was performed in a 4-20% polyacrylamide gradientgel. Proteins were transferred to a nitrocellulose filter andsequentially reacted with HuMAb L92 and peroxidase conjugated goatanti-human IgM antibody (Boehringer Mannheim, Ind.). Colorimetricreactions were performed with 4-chloro-1-napthol as the substrate.

In the resulting blot, a discrete band is seen in each of the celllysates (FIG. 1A). In a control experiment, an unrelated, purified humanIgM was tested against the same cell lysates (FIG. 1B). No correspondingband was detected on the control gel. The molecular weight of thereactive protein was estimated as 43 kD based on its mobility in theSDS-PAGE. Cell lysates obtained from M14 melanoma cells grown in humanserum media exhibited the same band, indicating that the protein was notderived from FCS in the culture media.

ELISA Analysis

Peptide ELISA was performed in Reacti-Bind™ Plates (PIERCE), asdescribed in the manufacturer's instructions. Briefly, synthetic peptidewas dissolved in 0.1M Sodium phosphate, 0.15M NaCl pH 7.2 and added toELISA plates (100 μl/well) by overnight incubation at 4° C. ELISA wasthen performed with HuMAb L92 or L612 using peroxidase conjugated goatanti-human IgM antibody. The color was developed with o-phenylenediamine(OPD, Sigma USA) and read on an ELISA reader at 490 nm.

EXAMPLE II Immunoprecipitation

Indirect immunoprecipitation of proteins labeled in vivo with ³⁵ Smethionine was performed in order to identify protein molecules reactiveto HuMAb L92. Briefly, M14 melanoma cells were cultured in RPMI 1640media supplemented with 10% FCS in T25 culture flasks (Costar) untilthey reached 70% confluence. The cells were then washed twice with amedia preparation consisting of methionine deficient RPMI 1640 media(Sigma R7130) dissolved in 1 L of double distilled water, to whichglutamic acid (0.02 g/L), lysine (0.04 g/L), leucine (0.05 g/L), and 2g/L of sodium bicarbonate were added. Two ml of this preparation,supplemented further with 10% dialyzed fetal calf serum (Sigma), wereadded to the flasks, which were subsequently incubated with 1.85 MBq of³⁵ S-methionine (Amersham) for 4 hrs. After harvesting and washing twicewith PBS, the cells were lysed with 100 ul of lysis buffer consisting of50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.02% sodium azide, 0.01% SDS, 100ug/ml phenylmethyl sulfonyl fluoride (PMSF), 1% NP-40, and 0.5% sodiumdeoxycholate.

Forty microliters of the cell lysate was diluted to 300 ul with lysisbuffer. The diluted lysate was pre-cleaned by incubation with 20 ul ofgoat anti-human IgM-biotin (Boehringer) for 1 hr on ice and thenincubated with 100 ul of Streptavidin-agarose (Sigma) for an additional1 hr. The supernatant was collected via centrifugation and divided into3 aliquots. One aliquot was incubated with 10 ug of HuMAb L92. Thesecond aliquot was incubated with HuMAb L612 (unrelated antibody), andthe third aliquot was incubated in the absence of antibody for 1 hr onice. Ten microliters of anti-human IgM-biotin was added to each of thethree aliquots and incubated for an additional 1 hr. Ten microliters ofStreptavidin-agarose was then added to each aliquot and incubated foranother 1 hr. The antigen-anti-IgM-biotin-Streptavidin-agarose complexwas pelleted via centrifugation and washed three times with 1 ml oflysis buffer followed by washing with 1 ml of 10 mM Tris-HCl (pH 7.0)and 1% NP-40. Immunoprecipitates were analyzed in 4-20% polyacrylamidegradient SDS gels under reducing conditions. The gels were treated withAmplify™ (Amersham) and processed for autoradiography.

EXAMPLE III Construction of the Expression Library

To isolate the cDNA clones that encode the HuMAb L92 immunopositiveprotein, a cDNA expression library from M14 cells was constructed usingthe λgt11 recombinant phage system. Approximately 6×10⁵ phage werescreened and 2 positive clones were obtained, one of which was chosenfor subsequent analysis.

Briefly, the M14 cells were cultured in RPMI-1640 media supplementedwith 10% FCS and antibiotic (GIBCO, N.Y.). Total RNA was isolated from5×10⁸ M14 cells using guanidine isothiocyanate (Chirgwin, J. M. et al.,1979). Poly A⁺ RNA was prepared using the Poly A Tract™ mRNA IsolationSystem (Promega). cDNA was prepared using the Copy™ Kit (Invitrogen).After ligation of an EcoRI adaptor to both ends, cDNA was inserted intothe EcoRI site of λgt11 phage DNA. In vitro packaging was performedusing GigaPak II Gold (Stratagene). The library was amplified once onsolid media before screening.

EXAMPLE IV Screening of the cDNA Library

E. coli Y1090 was cultured at 37° C. in LB broth supplemented with 0.2%maltose and 0.5% MgSO₄ until the OD600 reached 0.5. Cells were collectedand suspended in half of the original volume in 10 mM MgSO₄. Cells wereinfected by the phage library of Example III and plated on LB agarplates (approximately 20.000 plaques per 150 mm plate). The plates wereincubated for 3.5 hrs at 42° C. After placing nitrocellulose filterswhich had been immersed in 10 mM IPTG on the plates, they were incubatedfor an additional 4 hrs at 37° C. The filters were subsequentlyimmunostained by HuMAb L92 and peroxidase conjugated goat anti-human IgMantibody. The coloring reaction was carried out with 4-chloro-1-naphtol.The plaques which corresponded to a positive signal on the filters wereremoved from the agar plates and eluted to prepare the phage stock. Thescreening procedure was repeated until an homogeneous population ofimmunopositive recombinant bacteriophage was obtained.

EXAMPLE V Sequencing the cDNA Insert

To characterize the cloned DNA, an attempt was made to subclone theinsert DNA into the EcoRI site of pBluescript II (Stratagene). However,the insert DNA could not be isolated with EcoRI digestion. Therefore,whole phage DNA was used as the template to determine the DNA sequence.A partial nucleotide sequence of the cDNA insert in the recombinant λt11phage was determined using the Double Stranded DNA Cycle SequencingSystem (BRL, Gaithersburg, Md.).

As shown in FIG. 4, this clone has a one base deletion at the EcoRIsite, which made it impossible to subclone the insert into the EcoRIsite of the plasmid. Although the insert DNA of this clone is 1.4 kbp,it has only a 10 amino acid open reading frame after the EcoRI linkersequence. A search for homology between the open reading frame sequenceof the insert and the reported DNA sequences in GeneBank, however,revealed no significant homology.

EXAMPLE VI Bacterial Expression of the Protein-Peptide Fusion

To determine the antigenicity of the cloned cDNA, it was expressed as afused protein with β-galactosidase in the E. coli lysogen system and therecombinant protein was analyzed by a Western blot (FIG. 3). Fiftymicroliters of recombinant phage lysogen which had been culturedovernight at 32° C. were inoculated in 5 ml of LB/ampicillin liquidmedia and cultured at 32° C. with shaking. When the OD600 reached 0.5,the temperature was shifted to 42° C. for 10 min. Isopropylβ-D-thiogalactoside (IPTG) was added to the culture with a finalconcentration of 1 mM. Cells were cultured at 37° C. for 2 hrs,harvested via centrifugation, and analyzed by SDS-PAGE.

HuMAb L92 reacted with a band at 106 kD in the Western blot (FIG. 3,Lane 1). The lysate obtained from a control experiment (without IPTGinduction) did not show this band (Lane 2). In lane 3, β-galactosidaseshows a clear band in CBB staining, however, this band also does notbind HuMAb L92. These data indicate that the amino acids derived fromthe cDNA portion of the β-galactosidase-peptide fusion exhibitantigenicity against HuMAb L92.

EXAMPLE VII Antibody Recognition of a Cloned Oligopeptide

An oligopeptide was synthesized based on the deduced amino acid sequenceof the cDNA and it Was tested for its ability to inhibit HuMAb L92binding to the recombinant protein-peptide fusion and to M14 celllysates. HuMAb L92 was pre-incubated with 500×molar excess of a 14 aminoacid peptide (DSRPQDLTMKYQIF, seq id no:2). This peptide sequencecontains the amino acid sequence derived from the cDNA open readingframe and an additional four amino acids derived from the EcoRI adaptor.HuMAb L92 that was not preincubated with the 14 amino acid peptideformed a discrete band with both the protein-peptide fusion and M14 cellextracts at 106 kD and 43 kD, respectively (FIG. 5A). However, HuMAb L92that was preincubated with the peptide exhibited a significant reductionin reactivity and formed only a faint band on the Western blot (FIG.5B). The 10 amino acid peptide (QDLTMKYQIF, seq id no:1) whichcorresponds to the cDNA open reading frame showed similar bindinginhibition of HuMAb L92 to the protein-peptide fusion and M14 celllysates in a Western blot analysis. The direct binding reaction of HuMAbL92 to the 10 amino acid peptide was also examined by an ELISA assay. Astrong and specific reaction was demonstrated using 1 ug of peptide perwell (FIG. 6). These results indicate that the 10 amino acidoligopeptide has antigenicity to HuMAb L92.

In order to further determine the minimum length required for theantigenic epitope of HuMAb L92, several GST-fused peptides that weretruncated from the 10 amino acid immunoreactive peptide sequence (seq idno:1) were prepared using synthetic oligonucleotides and pGEX-2T as thevector. While the truncation of 1 amino acid residue from the C-terminusdid not affect the binding of HuMAb L91 (FIG. 14B, Lane 3), the removalof 2 C-terminal amino acid residues completely deleted antibody binding(lane 4). The deletion of 6 amino acid residues from the N-terminus ofthe 10 amino acid peptide did not affect the binding of HuMAb L92 (Lane7), but the removal of 7 N-terminal amino acids again deleted antibodybinding (Lane 8). These results demonstrate that the minimum antigenicepitope of HuMAb L92 is a 4 amino acid peptide (Lys-Tyr-Gln-Ile, seq idno:8).

EXAMPLE VIII Localization of the 43 kD Protein in Melanoma Cells

To determine whether the 43 kD protein is expressed on the cell surfaceor remains inside the cell, HuMAb L92 was absorbed with intact M14 cellsand then allowed to react to the recombinant protein-peptide fusion andto the 43 kD protein in a Western blot analysis (FIG. 7). The stainingintensities of the 43 kD protein of M14 cell lysate and of the 106 kDprotein of #810 lysogen were only slightly reduced after absorption ofthe antibody with intact M14 cells. This result suggests that the 43 kDprotein is not expressed in significant amounts on the M14 cell surface.However, subsequent analysis of other melanoma cell lines has shown thatthe density of the 43 kD protein on the cell surface varied widely amongcell lines. For example, the UCLASO M25 cell line (Ravindranath M. H. etal., 1989) expressed the highest degree of the 43 kD protein on the cellsurface among 20 human melanoma cell lines tested.

In situ hybridization

A synthetic oligodeoxynucleotide sequence complementary to the mRNA for#810 peptide, seq id no:2 was end-labeled with digoxigenin and used todetect mRNA in a panel of cells. In situ hybridization was performed asdescribed (Morisaki et al., 1992). The cells fixed on glass slides wereprehybridized for 1 h at 42° C. in a solution containing deionizedformamide, 20×standard saline citrate, Denhardt's solution,heat-denatured sheared herring sperm DNA, yeast transfer RNA and dextransulfate. #810 antisense probe (5'-AAA GAT CTG ATA TTT CAT AGT CAG ATCCTG-3', seq id no:3, Molecular Biology Institute, UCLA School ofMedicine, Los Angeles, Calif.) was tail-labeled with digoxigenin-11-dUTP(Boehringer Mannheim, Indianapolis, Ind.) using the DNA tailing kit(Boehringer Mannheim). A negative control was carried out by using #810sense probe which was complementary to the test (antisense) probe in thereaction. A 27-mer oligonucleotide probe specific to human fibroblastβ-actin (5'-GAC GAC GAG CGC GGC GAT ATC ATC ATC-3', seq id no:4,Clontech Laboratories, Inc., Palo Alto, Calif.) was used for a positivecontrol. Digoxigenin-labeled probe was placed on the cells and incubatedat 42 ° C. overnight. Cells containing #810 peptide mRNA were detectedusing the Genius nonradioactive nucleic acid detection kit (BoehringerMannheim). Slides were incubated with 2% normal sheep serum and 0.3%Triton X-100 at room temperature for 30 min. Anti-digoxigenin antibodywas applied to the cells for 3 h at room temperature. A solutioncontaining nitroblue tetrazolium, X-phosphate solution and Levamisolewas placed on the cells at room temperature until they developed asatisfactory color (2-5 h).

In situ hybridization showed that mRNA for #810 peptide was commonlyexpressed in melanomas, non-melanoma tumors and also in normallymphocytes (Table 1). FIG. 8 shows a representative melanoma cell line.Hybridization with #810 antisense probe revealed dense staining at asingle cell level whereas the use of a sense probe exhibited nostaining, demonstrating the probe specificity. Western blotting revealedthat 43 kD protein including the sequence of #810 amino-acid sequencecould be detected in the same cells that expressed #810 mRNa and thusthe presence of this protein was not specific to melanomas. However, itslocalization varied among cell types; a high density of 43 kD proteinwas located on the cell surface of certain cell lines (M12, M25, SHN,etc.) while it was not detected on the surface of other kinds (M14, M24,normal lymphocytes, etc.), which results were also proven by antibodyabsorption assays using those cells. The relations between the histologyof those cells and the intracellular localization of the 43 kD proteinhave not been clarified yet.

                  TABLE 1                                                         ______________________________________                                        Expression of peptide antigen #810 (QDLTMLYQIF, seq id                        no:1) and 43 kD protein by tumors and normal cells.                                         mRNA for                                                                      #810 peptide                                                                           43 kD protein                                                        in situ  Western cell surface                                                 hybridization.sup.a                                                                    blot.sup.b                                                                            IA assay.sup.c                                 ______________________________________                                        melanoma M10    +          +       +                                               M12        ++         +       +++                                             M14        +          +       -                                               M15        +          +       -                                               M24        +          +       -                                               M25        ++         +       ++++                                            M101       +          +       +                                               M111       +          +       -                                               M112       +          +       -                                          neuroblastoma SHN                                                                             +          +       ++++                                       colon cancer SW48                                                                             +          +       ++                                              SW480      +          +       +                                          gastric cancer MKN28                                                                          +          +       -                                                MKN45     +          +       ++                                         lung cancer 130 +          +       +                                               135        +          +       -                                          breast cancer 645                                                                             +          +       -                                          erythroleukemia K562                                                                          +          +       -                                          B cell lymphoma L14                                                                           +          +       -                                          normal cells in peripheral blood                                              monocytes       +          +       -                                          T cells         +          +       -                                          B cells         +          +       -                                          erythrocytes    ND.sup.d   -       ND                                         phytohemagglutinin- stimulated                                                blood lymphocytes                                                                             +          +       -                                          ______________________________________                                         .sup.a Expression of #810 mRNA was analyzed by in situ hybridization.         .sup.b Presence of 43 kD protein was analyzed by Western blot using L92       antibody.                                                                     .sup.c Cell surface expression of 43 kD protein was analyzed by               immunoadherence (IA) assay using L92. , target cells forming erythrocyte      rosettes are <5% of total; ± , 5-10%; +, 10-20%; ++, 20-50%; +++,          50-75% and ++++, >75%.                                                        .sup.d Not done                                                          

EXAMPLE IX A Peptide Vaccine

A peptide which comprises as part of its amino acid sequence, a sequencein accordance with seq id no:1 may be clinically very important as aneffective vaccine in inducing anti-tumor humoral and cell-mediatedimmune responses in cancer patients. The inventors' results have shownthat the synthetic peptide has the ability not only to stimulate theproliferation of lymphocytes of melanoma patients who received melanomacell vaccine, but also to induce cytotoxic T cells in vitro againstautologous melanoma cells.

As is well known in the art, a given polypeptide may vary in itsimmunogenicity. It is often necessary therefore to couple the immunogen(e.g., a polypeptide of the present invention) with a carrier. Exemplaryand preferred carriers are keyhole limpet hemocyanin (KLH) and humanserum albumin. Other carriers may include a variety of lymphokines andadjuvants such as INF, IL2, IL4, IL8 and others.

Means for conjugating a polypeptide to a carrier protein are well knownin the art and include glutaraldehyde,m-maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimyde andbis-biazotized benzidine. It is also understood that the peptide may beconjugated to a protein by genetic engineering techniques that are wellknown in the art.

As is also well known in the art, immunogenicity to a particularimmunogen can be enhanced by the use of non-specific stimulators of theimmune response known as adjuvants. Exemplary and preferred adjuvantsinclude complete BCG, Detox, (RIBI, Immunochem Research Inc.) ISCOMS andaluminum hydroxide adjuvant (Superphos, Biosector).

The preparation of vaccines which contain peptide sequences as activeingredients is generally well understood in the art, as exemplified byU.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792;and 4.578,770, all incorporated herein by reference. Typically, suchvaccines are prepared as injectables, either as liquid solutions orsuspensions: solid forms suitable for solution in, or suspension in,liquid prior to injection may also be prepared. The preparation may alsobe emulsified. The active immunogenic ingredient is often mixed withexcipients which are pharmaceutically acceptable and compatible with theactive ingredient. Suitable excipients are, for example, water, saline,dextrose, glycerol, ethanol, or the like and combinations thereof. Inaddition, if desired, the vaccine may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, pH buffering agents,or adjuvants which enhance the effectiveness of the vaccines.

The peptide may be formulated into the vaccine in a neutral or saltform. Pharmaceutically acceptable salts, include the acid addition salts(formed with the free amino groups of the peptide) and those which areformed with inorganic acids such as, for example, hydrochloric orphosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups mayalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, 2-ethylamino ethanol,histidine, procaine, and the like.

The vaccine will be administered in a manner compatible with the dosageformulation, and in such amount as will be therapeutically effective andimmunogenic. The quantity to be administered depends on the subject tobe treated, including, e.g., the capacity of the individual's immunesystem to respond. Precise amounts of active ingredient required to beadministered depend on the judgment of the practitioner. Suitableregimes for initial administration and booster shots are also variable,but are typified by an initial administration followed by subsequentinoculations or other administrations.

The manner of application may be varied widely. Any of the conventionalmethods for administration of a vaccine are applicable. These arebelieved to include oral application in a solid physiologicallyacceptable base or in a physiologically acceptable dispersion,parenterally, by injection or the like. The dosage of the vaccine willdepend on the route of administration and will vary according to thedetermination of the practitioner for each particular patient.

EXAMPLE X Diagnostic Immunoassays and Skin Tests

Immunoassays that measure the degree of specific humoral and cellularimmune responses in patients during active, specific immunotherapy canbe developed in light of the present disclosure. The peptides andprotein of this invention can be evaluated for their in vivoimmunogenicity and therapeutic efficacy in immunotherapy. A variety ofhumoral and cell-mediated assays have been developed to define thesystem with the greatest ability to predict clinical responses. Thehumoral assays include the immune adherence, membraneimmunofluorescence, FACS analysis, ELISA, and radioimmunoassays.

In light of the present disclosure, the synthetic peptide antigens maybe used to establish more accurate serologic assay systems and skintests that have the ability to predict immune and clinical responsesinduced by these newly discovered antigens.

EXAMPLE XI Use of the cDNA to Isolate the Full Length Gene

Full-length peptide analysis of the antigenic protein is important inorder to determine the location of the antigen epitope, to search othersites for cross reactive antigens within the 43 kD protein, and toevaluate its biological function and pathogenic significance for thedisease. The sequence of the cloned cDNA #810 is useful as a primer or aprobe to isolate the full length gene encoding the 43 kD protein. Thetechniques to accomplish the isolation of the full length gene are wellknown in the art.

For example, a genomic library could be constructed by well knowntechniques and screened with the cDNA clone #810. The library would beseparated, for example by polyacrylamide gel electrophoresis, or agarosegel electrophoresis and then transferred to a filter such as anitrocellulose filter. The clone #810 would then be labeled with ³² P byenzymatic labelling with polynucleotide kinase, for example. The clonecould also be radioactively labeled by nick translation or in apolymerase chain reaction that included radiolabeled nucleotides.Alternatively, the probe could be labeled with a fluorescent marker suchas biotin or any fluorophore. Such labelling techniques are well knownin the art.

The labeled probe would then be hybridized to the denatured DNA on thefilter and washed under increasingly stringent conditions, incrementallyhigher temperatures for example, until the positive clones can beidentified by autoradiography or by fluorescence. These positive cloneswould then be rescreened and sequenced to determine the full genesequence encoding the 43 kD protein.

The full protein could then be expressed in an E. coli strain, forexample, and used for further analysis. It is understood that theprotein could also be truncated or altered by site directed mutagenesis,for example and that such altered proteins or partial sequences wouldalso fall within the scope of the present invention.

EXAMPLE XII CTL Response to the Antigen

Cytotoxicity assays

A standard ⁵¹ Cr release assay was performed as previously described(Hayashi et al., 1992). Melanoma cell lines were harvested and labeledwith 100 μCi⁵¹ Cr (Amersham, Arlington Heights, Ill.) for 2 h at 37° C.and used as targets. The labeled targets were washed and resuspended inculture medium and seeded in round-bottom 96 well microtiter plates at5×10³ cells/well and then effector cells were added. All assays (200μl/well) were carried out in triplicate. After 6 h of incubation, 100 μlof supernatants were collected and counted. % specific cytolysis wascalculated as follows: % specific cytolysis=100×(experimentalrelease-spontaneous release/5% Triton X release-spontaneous release).

For peptide experiments, BCL were pulsed with a peptide at 10 μM,simultaneously labeled with 100 μCi⁵¹ Cr for 2 h at 37° C. and were usedas targets. 17% lysis was set as the threshold for defining a positiveor negative response at an E:T of 80:1. This value was selected becauseit was 3SD above the mean lysis obtained from two negative controls:PBMC from 19 control normal donors tested on autologous BCL pulsed withpeptide #810, seq id noP:1, (3SD above mean=16.9%); and PBMC from 19melanoma patients tested on autologous BCL with medium only or pulsedwith an irrelevant decapeptide (3SD above mean=16.5%).

Representative data of 19 patients and 19 control normal donors areshown in FIG. 9A. At 0, 4 and 8 weeks, a patient's PBMC show positivecytolyses (a 17% at E:T of 80:1) of autologous #810, seq id no:1--pulsedBCL while those of a normal donor do not. Lysis of autologous BCLwithout peptides did not exceed 7% by any effector. Mean cpm±SD oftriplicate cultures are shown in FIG. 9B. At 4 and 8 weeks, values ofthe patient's PBMC with #810, seq id no:1 peptide 0.001-20 μM aresignificantly higher than those without #810, seq id no:1 (p<0.005) andmaximal SIs were 2.71 and 2.87 at 4 and 8 weeks respectively, whereassignificant proliferative response to #810, seq id no:1 is not observedbefore vaccination (0 weeks) and in a control donor.

Proliferation assays

PBMC (1×10⁵ cells/well) were seeded into 96-well plates and peptideswere added at desired concentrations in a total 200 μl/well of culturemedium. Tritiated thymidine ( ³ H!TdR) (0.5 μCi/well, Amersham ArlingtonHeights, Ill.) was added during the last 18 h of a 4 day culture period.Cultures were set up in triplicate and harvested with a Brandel cellharvester and then counted in a Beckman scintillation counter.Stimulation index (SI) was calculated as follows: mean cpm of triplicatecultures with peptide!\ mean cpm without peptide!. Maximal SI amongvarious concentrations≧2.0 was defined as a positive response to peptide#810, seq id no:1 because it was 3SD above the mean of maximal SIobtained from two negative controls: PBMC from 19 control donors testedon #810, seq id no:1(3SD above mean=1.97); and PBMC from 19 melanomapatients tested on an irrelevant decapeptide (3SD above mean=1.37).

                  TABLE 2                                                         ______________________________________                                        Summary of peptide #810-specific cytotoxicity and                             proliferative response of PBMC from vaccinated melanoma patients.             cytotoxicity.sup.a   proliferative response.sup.b                             mean ± SD    positive mean ± SD                                                                             positive                                  of % lysis      response of maximal response                                  (E:T = 80.1)    (%)      SI         (%)                                       ______________________________________                                        vaccinated                                                                            0w 19.85 ± 4.80.sup.d                                                                  13 (68.4)                                                                              1.62 ± 0.31.sup.d                                                                   3 (15.8)                                melanoma                                                                              4w          15 (78.9)                                                                              2.43 ± 0.55.sup.d,e                                                                 12 (63.2)                                       40.25 ± 9.13.sup.d,e                                               patients.sup.c                                                                        8w          16 (84.2)                                                                              2.50 ± 0.60.sup..sup.d,e                                                            13 (68.4)                                       38.70 ± 8.76.sup.d,e                                               (n = 19)                                                                              positive    16 (84.2)         13 (68.4)                                       response                                                                      either pre or post                                                            vaccination                                                                   enhanced    15 (78.9)         12 (63.2)                                       response.sup.f                                                                post                                                                          vaccination                                                           control 6.10 ± 3.60                                                                            2 (10.5) 0.98 ± 0.33                                                                         0 (0)                                   (n = 19)                                                                      ______________________________________                                         .sup.a,b Cytotoxicity against autologous BCL pulsed with #810 and             proliferative response to #810 were evaluated as described above.             .sup.c Melanoma patients were immunized with MCV and were tested before       (0w) and at monthly intervals (4, 8w) after the initiation of vaccination     .sup.d Significant difference vs values of control (p < 0.005).               .sup.e Significant difference vs values of 0w (p < 0.005).                    .sup.f Number and % of patients are shown whose responses to peptide #810     were significantly increased either at 4 or 8w compared to 0w (p < 0.05).

PBMC from vaccinated melanoma patients were assayed for peptide #810recognition and the results were compared among various phases (0, 4,8w) and also compared with those of control normal donors. In sixteen of19 melanoma patients (84.2%), PBMC recognized and lysed autologous BCLpulsed with #810 peptide, seq id no:1, either before or aftervaccination while PBMC from only two of 19 normal donors (10.5%) showedsuch cytotoxicity (Table 2, FIG. 9A). This cytotoxicity was specific to#810, seq id no:1, since PBMC from patients and normal donors failed tolyse autologous BCL without peptides or pulsed with a decapeptide ofirrelevant amino-acid sequence. Such #810, seq id no: 1--specificcytotoxicity of melanoma patients was significantly increased aftervaccination in 15 patients (78.9%). In most cases that activity was thehighest at 4 weeks and rather reduced at 8 weeks, however, it was stillabove the prevaccination level.

PBMC from melanoma patients also showed proliferative response to #810,seq id no:1 although the magnitude of response was 'weaker than that ofcytotoxicity assays (FIG. 9B). In 13 patients (68.4%), ³ H!TdR uptake ofpeptide-stimulated PBMC was increased more than two-fold over thosewithout #810, seq id no:1, and such proliferative response wassignificantly enhanced after vaccination in 12 patients (63.2%). Thatresponse was also specific to #810, seq id no:1 since that was notobserved when a patient's PBMC were incubated with an irrelevantdecapeptide.

Those results suggest that PBMC only from melanoma patients may belatently sensitized in vivo with #810, seq id no:1, even beforevaccination and cellular immune responses to this peptide may berecalled in vitro, indicating that autologous melanoma cells mayspecifically present #810, seq id no:1. The augmentative effect ofvaccination suggests that allogeneic melanoma cells of MCV may alsopresent #810, seq id no:1, to patients' lymphocytes and enhance specificresponses to this peptide. In contrast, PBMC of donors may not be invivo sensitized with #810, seq id no:1, though it does commonly exist inbenign somatic cells.

EXAMPLE XIII Effect of in vitro Restimulation with Peptide of SEQ IDNO:1

PBMC (3×10⁶ well) from melanoma patients 4 weeks after MCV immunizationwere stimulated with #810 peptide, seq id no:1, at 10 μM in 2ml culturemedium in 24-well plates at 37° C. After 5 days of incubation, cellswere harvested and assayed for cytotoxicity.

PBMC from patient A 4 weeks after vaccination were in vitro restimulatedwith #810, seq id no:1, and were assayed for cytolyses of autologous#810, seq id no:1--pulsed BCL and melanoma MA targets (FIG. 10). □ and ∘indicate cytotoxicity of control PBMC cultured with medium only. Lysisof autologous BCL without peptides or pulsed with an irrelevantdecapeptide did not exceed 8% at an E:T of 80:1 by either effector.Lysis of K562 by #810, seq id no:1--restimulated PBMC and control at E:Tof 80:1 were 24.0 and 24.5%, respectively. Data are representative ofthree separate experiments.

Cold Target Inhibition Assays

Unlabeled cold target cells (50 μl) were seeded in 96-well plates atappropriate concentrations. Effector cells (100 μl) were added into thewell and incubated for 1 h at 37° C. before the addition of ⁵¹Cr-labeled hot targets at desired cold:hot target ratios.

PBMC from patient A were restimulated with #810, seq id no:1 similarlyto FIG. 10 and assayed for cytolyses of autologous ⁵¹ Cr-labeled #810,seq id no:1--pulsed BCL (FIG. 11A) and melanoma MA (FIG. 11B) at E:T of20:1. Unlabeled cold target cells were added at indicated ratios. Lysisof BL without peptides was 3.0% at E:T of 20:1. Data are representativeof four separate experiments.

#810, seq id no:1--restimulated PBMC from vaccinated patient A revealedmore than a 3-fold increase in cytotoxicity against autologous #810, seqid no:1--pulsed BCL (67.3%, E:T=40:1) compared to a control culturedwithout peptide (18.0%) (FIG. 10). Such cytotoxicity was #810, seq idno:1--specific and different from non-specific lymphokine-activatedkiller (LAK) activity since killing activity against K562 (LAK target)or autologous BCL without peptides or pulsed with an irrelevantdecapeptide was not increased. Furthermore, this #810, seq idno:1--restimulation simultaneously enhanced killing activity againstautologous melanoma cells. These results indicate that the identical#810 antigen, seq id no:1, may possibly be presented on the surface ofautologous melanoma cells similarly to #810, seq id no:1--pulsed BCL.That possibility was explored by cold-target inhibition tests. Coldautologous melanoma cells inhibited lysis of autologous #810, seq idno:1--pulsed BCL, although less efficiently than unlabeled #810, seq idno:1--pulsed BCL (FIG. 11A). Conversely autologous #810, seq idno:1--pulsed BCL completely blocked lysis of autologous melanomas (FIG.11B). These data indicate that #810 antigen, seq id no:1, may bepresented on the surface of melanoma cells and can be recognized as atarget antigen of cytolysis.

Antibody Inhibition Assays

#810, seq id no:1--restimulated PBMC from patient A were assayed forcytolyses of autologous #810, seq id no:1--pulsed BCL and melanoma MA atE:T of 40:1 in the presence or absence of antibodies. A finalconcentration of each antibody was 10 μg/ml. Lyses of autologous BCLwithout peptides and pulsed with an irrelevant decapeptide were 5.4 and5.2%, respectively. Data are representative of four separateexperiments.

EXAMPLE XIV HLA Restriction of Peptide-Specific CTL

HLA typing

To identify the class I determinants that restrict the recognition ofpeptide #810, seq id no:1, a series of HLA-typed melanoma patients weretested (Table 3). PBMC from patients were used for HLA typing by acomplement-mediated microcytotoxicity assay in Dr. Paul Terasaki'slaboratory (UCLA School of Medicine, Los Angeles, Calif.).

Restriction of Recognition

As it has often been demonstrated that HLA-A antigens are importantrestrictive elements for CTL recognition of melanomas (Kawakami et al.,1992), HLA-A epitopes among class I antigens were selected. InHLA-A2+patients, A, B and C., in vitro restimulation with #810, seq idno:1, succeeded in the enhancement of CTL activity against bothautologous #810, seq id no:1--pulsed BCL and melanoma targets. Bycontrast, #810, seq id no:1--restimulated PBMC from A2(-) patient G didnot show such CTL activity though this patient shared A29 with patientB. These suggest HLA-A2 may serve as a restricting element for #810, seqid no:1 recognition. Among A2(-) patients, #810, seq idno:1--restimulated PBMC from patient D (A11, 30) and E (A28, 31)appeared to recognize #810, seq id no:1, while those from patient F(A24,-), G (A29,-) and H (A24, 32) did not. HLA-B antigens did notappear to play a significant role in #810, seq id no:1--recognition;#810, seq id no:1n--restimulated PBMC from patient A (HLA-B7+) andpatient B (B44+) recognized peptide #810, seq id no:1, while those frompatient H (B7&44+) did not. The present results indicate that A2 and Allmay function as such restricting elements for #810, seq id no:1,recognition in patient A-D while A24 may not (patient F and H).

To verify this possibility, #810, seq id no:1--restimulated PBMC frompatient B (A2+) and D (A11+) were tested for cytolyses of autologous andallogeneic HLA-A-matched or mismatched BCL pulsed with #810, seq idno:1(FIG. 13). #810, seq id no:1--restimulated PBMC from A2+ patient Bcould lyse allogeneic A2+ BCL pulsed with #810, seq id no:1, whereasthey failed to lyse A2(-) targets. Similarly #810, seq idno:1--restimulated PBMC from A11+ patient D could only lyse A11+ BCLpulsed with #810, seq id no:1. BCL targets without #810, seq id no:1were not lysed by those effectors, indicating that the lysis waspeptide-specific and not allo-reactive. These results suggest thatHLA-A2 and A11 can serve as restricting elements for #810, seq id no:1,recognition. Such restriction was further evaluated against HLA-matchedor mismatched melanoma targets (Table 4). In A2+ patients (A, B), #810,seq id no:1--restimulation enhanced the cytotoxicity against A2+melanoma targets while such enhancement was not observed against A2(-)melanoma targets. Similar results were obtained in A11+ patient D. Thoseresults suggest that melanomas may present #810 antigen, seq id no:1, toCTL in association with class I molecules, at least with HLA-A2 and A11,and also indicate that #810, seq id no:1, may function as a CTL epitopeof melanoma.

                  TABLE 3                                                         ______________________________________                                        HLA restriction of peptide recognition by #810, seq id                        no:1 -restimulated PBMC.                                                                   % specific cytolysis.sup.a vs                                    HLA            autologous    autologous                                       patients                                                                             A       B       #810-pulsed BCL                                                                           melanoma                                   ______________________________________                                        A      2,3     7,8     64.3   (18.0) 55.1  (5.4)                              B      2,29    44,-    25.9   (13.5) 17.4  (5.9)                              C      2,28    27,58   31.8   (14.9) 24.1  (3.2)                              D      11,30   18,70   29.7   (19.0) 37.5  (9.6)                              E      28,31   39,60   30.3   (21.4) 12.0  (8.0)                              F      24,-    48,53   11.0   (10.0) 3.0   (3.0)                              G      29,-    49,-    3.4    (7.2)  3.0   (4.0)                              H      24,32   7,44    0      (0)    11.0  (10.4)                             ______________________________________                                         .sup.a PBMC from vaccinated patients were restimulated in vitro with or       without peptide #810, seq id no:1, for 5d and were analyzed for               cytotoxicity at E:T of 40:1. Values in parentheses indicate cytotoxicity      of control PBMC cultured without peptide. Lysis of autologous BCL without     peptides or pulsed with an irrelevant decapeptide did not exceed 7% at        this ratio by any effector. Data of each patient are representative of at     least three separate experiments.                                        

                                      TABLE 4                                     __________________________________________________________________________    Cytotoxicity of #810, seq id no:1 -restimulated PBMC                          against autologous and allogeneic melanoma targets.                           Target                                                                              M10 M24 M101                                                                              MA  MB  MD  MN  MP  K562                                    HLA-A 24,33                                                                             11,33                                                                             2,29                                                                              2,3 2,29                                                                              11,30                                                                             1,2 2,9 --)                                     __________________________________________________________________________    patient A.sup.b                                                               control                                                                             9.8 9.1 8.6 5.4 9.5 3.0 6.4 7.4 15.1                                    HLAA2,3                                                                             9.3 1.6 37.8                                                                              55.1                                                                              23.6                                                                              4.0 20.2                                                                              18.0                                                                              16.0                                    #810                                                                          patient B                                                                     control                                                                             8.2 11.6                                                                              9.1 8.6 5.9 6.0 9.5 11.1                                                                              15.7                                    HLAA2,29                                                                            9.2 11.9                                                                              35.1                                                                              31.4                                                                              17.4                                                                              7.0 16.1                                                                              31.7                                                                              14.6                                    #810                                                                          patient D                                                                     control                                                                             8.2 9.0 3.1 11.7                                                                              4.9 9.6 6.3 8.7 17.7                                    HLAA11,30                                                                           7.1 37.4                                                                              2.0 8.0 5.0 37.5                                                                              6.9 9.8 16.1                                    #810                                                                          __________________________________________________________________________     .sup.a PBMC from vaccinated patients were restimulated in vitro with or       without peptide #810, seq id no:1, for sd and were analyzed for               cytotoxicity against a variety of melanoma targets at E:T of 40:1.            Parentheses indicate HLAA typing of a target or effector. Underlined          results denote autologous or allogeneic melanoma targets sharing HLAA         antigens with effectors. Cytotoxicity against K562 was tested as a            negative control. Data of each patient are representative of four separat     experiments.                                                                  .sup.b Patient A, B, D are bearers of melanoma MA, MB, MD, respectively. 

Representative data of three experiments are shown as % specificcytolysis at E:T of 40:1 against #810, seq id no:1--pulsed BCL targetsin FIG. 13. Effectors are #810, seq id no:1--restimulated PBMC ofpatient B (HLA-A2+) and D (HLA-A11+).

Parentheses of left margin indicate HLA-A or B antigens shared betweenthe effectors and targets. Each autologous and allogeneic target wasalso preincubated in media only or pulsed with an irrelevantdecapeptide, and was tested for lysis by the effectors. Lysis of thosecontrol targets did not exceed 5% by either effector.

EXAMPLE XV Effect of L92 on Cytolysis by #810, SEQ ID NO:1--Induced CTL

An attempt was made to block both autologous #810, seq id no:1--pulsedBCL and melanoma killing by monoclonal antibody L92, directed against#810, seq id no:1. Although this antibody showed specific bindingactivity to #810, seq id no:1, in a solid-phase peptide enzyme-linkedimmunosorbent assay (ELISA), it could neither bind substantially to a#810, seq id no:1--pulsed BCL nor melanomas of patient A in IA assays(Table 5). Similar results were obtained in flow cytometries using L92.This antibody also exhibited only slight inhibition of CTL lyses of bothtargets and inhibitory effect was not changed when antibodyconcentration was increased up to 50μg/ml. Thus antibody L92 could notdetect #810 antigen, seq id no:1, on the surface of target cellsalthough that antigenic epitope appeared to be associated with class Imolecules and recognized by CTL.

While the compositions and methods of this invention have been describedin terms of preferred embodiments, it will be apparent to those of skillin the art that variations may be applied to the composition, methodsand in the steps or in the sequence of steps of the method describedherein without departing from the concept, spirit and scope of theinvention. More specifically, it will be apparent that certain agentswhich are both chemically and physiologically related may be substitutedfor the agents described herein while the same or similar results wouldbe achieved. All such similar substitutes and modifications apparent tothose skilled in the art are deemed to be within the spirit, scope andconcept of the invention as defined by the appended claims.

                  TABLE 5                                                         ______________________________________                                        Inhibition of autologous #810, SEQ ID NO:1 -pulsed BCL                        and melanoma lyses by monoclonal antibody to #810 peptide.                             Target Cells                                                                  autologous   autologous                                                       #810-pulsed BCL                                                                            melanoma (MA)                                           Treatment.sup.a                                                                          IA assay.sup.b                                                                          % lysis.sup.c                                                                          IA assay                                                                              % lysis                                 ______________________________________                                        control              67.3             58.2                                    L92 (anti-#810)                                                                          ±      54.4     -       43.6                                    L612 (anti-GM.sub.3)                                                                     ND.sup.d  65.3     ND      54.3                                    anti-class I                                                                             ND        5.2      ND      8.0                                     ______________________________________                                         .sup.1 Target cells were preincubated for 1h with or without monoclonal       antibodies before the addition of effector cells. A final concentration o     each antibody in a 6hcytotoxicity assay was 10 μg/ml. L612 and             anticlass I antibodies were used as a negative and positive control,          respectively.                                                                 .sup.b IA assays were performed against those targets as described above.     .sup.c Effector cells were #810, seq id no:1 restimulated PBMC from           vaccinated patient A (HLAA2,3); E:T = 40:1. Lysis of autologous BCL           without peptides or pulsed with an irrelevant decapeptide did not exceed      8%. Data are representative of three separate experiments.                    .sup.d Not done.                                                         

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

Amagai M., Klaus-Kovtum V., Stanley J. R. Autoantibodies against novelepithelial cadherin in pemphigus vulgaris, a disease of cell adhesion.Cell 67:869-877, 1991.

Brown J. P., Hewick R. M., Hellstrom I., Hellstrom K. E., Doolittle R.F., Dreyer W. J. Human melanoma-associated antigen p97 is structurallyand functionally related to transferrin. Nature 296:171-173, 1982.

Brown J. P., Nishiyama K., Hellstrom I., Hellstrom K. E. Structuralcharacterization of human melanoma-associated antigen p97 in normal andneoplastic tissues. J Immunol 127:539-546, 1981.

Cahan L. D., Irie R. F., Singh R., Cassidenti A., Pauluson J. C.Identification of a human neuroectodermal tumor antigen (OFA-I-2) asganglioside GD2. Proc Natl Acad Sci USA 79:7629-7633, 1982.

Carey T. E., Lloyd K. O., Takahashi T., Travassos L. R., Old L. J. AUcell-surface antigen of human malignant melanoma. Solubilization andpartial characterization. Proc Natl Acad Sci USA 76;2898, 1979.

Challopadhyay P., Kaveri S.-V., Byars N., Starkey J., Ferrone S. Humanhigh molecular weight melanoma-associated antigen mimicry by ananti-idiotypic antibody: Characterization of the immunogenicity of theimmune response to the mouse monoclonal antibody IMel-1. Cancer Res51:6045-6051, 1991.

Cheresh D. A., Resifeld R. A., Varki A. P. O-acetylation ofdisialoganglioside GD3 gy human melanoma cells creates a uniqueantigenic determinant. Science 225:844-846, 1984.

Cheresh D. A., Varki A. P., Varki N. M., Stallcup W. B., Levine J.,Reisfeld R. A. A monoclonal antibody recognizes an O-acetylated sialicacid in a human melanoma-associated ganglioside. J Biol Chem259:7453-7459, 1984.

Chirgwin, J. M., Przybyla, A. E., McDonald R. J., Rutter, W. J.Isolation of biologically active ribonucleic acid from sources enrichedin ribonuclease. Biochem 18:5294-5299, 1979.

Dropcho E. J., Chen Y.-T., Posner J. B., Old L. J. Cloning of a brainprotein identified by autoantibodies from a patient with paraneoplasticcerebellar degeneration. Proc Natl Acad Sci, USA 84:4552-4556, 1987.

Erb K., Ditzel H., Weaver-Rasmussen J., Borup-Christensen P., JenseniusJ. C. Antigens recognized by two human monoclonal IgM anti-colon cancerantibodies, 16.88 and C-OU1 (B9165). Hum Antibod Hybridomas 2:215-221,1991.

Euhus D. M., Gupta R. K, Morton D. L. Association betweenallo-immunoreactive and xeno-immunoreactive subunits of a glycoproteintumor-associated antigen. Cancer Immunol Immunother 32:214-220, 1990.

Fontan E., Skalani-Jusforgues H., Fauve R. M. Immunostimulatory humanurinary protein. Proc Natl Acad Sci USA 89:4358-4362, 1992.

Fontan E., Skalani H., Fauve R. M. Macrophage-induced cytotoxicity andanti-metastatic activity of a 43-kDa human urinary protein against theLewis tumor. Int J Cancer 53:131-136, 1993.

Hayashi Y., Hoon D. S. B., Park M. S., Terasaki P. I., Foshag L. J. andMorton D. L. Induction of CD4+cytotoxic T cells by sensitization withallogeneic melanomas bearing shared or cross-reactive HLA-A. CellImmunol 139: 411-425, 1992.

Hayashibe K., Mishima Y., Ferrone S. Cloning and in vitro expression ofa melanoma-associated antigen immunogenic in patients with melanoma. JImmunol 147:1098-1104, 1991.

Hellstrom I., Hellstrom K. Melanoma vaccines-why and how. IN CutaneousMelanoma, C. M. Balch, A. N. Houghton, G. W. Milton, Sober A. J., andS-J. Song (Pub), J. B. Lippincott, PA, pp. 542-546, 1992.

Houghton A. N., Brooks H., Cote R. J., Taormina M. C., Oettgen H. F.,Old L. J. Detection of cell surface and intracellular antigens by humanmonoclonal antibodies. J Exp Med 158:53-65, 1983.

Irie R. F., Sze L. L., Saxton R. E. Human antibody to OFA-1, a tumorantigen, produced in vitro by EBV-transformed human B-lymphoblastoidcell lines. Proc Natl Acad Sci USA 79:5666-5670, 1982.

Irie R. F., Sze L. L., Saxton R. E. Human antibody to OFA-1, a tumorantigen produced in vitro by EBV-transformed human B-lymphoblastoid celllines. Proc Natl Acad Sci USA 79:5666-5670, 1982.

Irie R. F., Jones P. C., Morton D. L., Sidell N. In vitro production ofhuman antibody to a tumor-associated fetal antigen. Brit J Cancer44:262-266, 1981.

Irie R. F., Irie K., Morton D. L. A membrane antigen common to humancancer and fetal brain tissues. Cancer Res 36:3510-3517, 1976.

Kan-Mitchell J., Iman A., Kempf R., Taylor C. R., Mitchell M. S. Humanmonoclonal antibodies directed against melanoma tumor-associatedantigens. Cancer Res 46:2490-2510, 1986.

Kawakami Y., Zakut R., Topalian S. L., Stotter H. and Rosenberg S. A.Shared human melanoma antigens: recognition by tumor-infiltratinglymphocytes in HLA-A2.1-transfected melanomas. J Immunol 148:638-643,1992.

Kusama M., Kageshita T., Chen Z. J., Ferrone S. Characterization ofsyngeneic anti-idiotypic MAb to murine anti-human high molecularweight-melanoma-associated antigen (HMW-MAA). J Immunol 143:3844-3852,1989.

Larrick L. W., Gavilondo J. V., Coloma M. J., Fry K. E. Construction ofrecombinant therapeutic monoclonal antibodies. IN Therapeutic MonoclonalAntibodies, C. A. K. Borrebaech and L. W. Larrick (eds), Stockton Press,New York, pp. 17-35, 1990.

Li J., Henn M., Oratz R., Bystryn J. -C. The antibody response toimmunization to a polyvalent melanoma antigen vaccine. Clin Res 38:660A,1990.

Livingston P. O., Natoli E. L., Calves M. J., Stockert E., Oettgen H.F., old L. J. Vaccines containing purified GM2 ganglioside elicit GM2antibodies in melanoma patients. Proc Natl Acad Sci USA 84:2911-2915,1987.

Livingston P. O. Immune responses to melanoma vaccines: Focus ongangliosides. 35th Annual Clinical Conference Proceedings, M. D.Anderson Cancer Center, Houston, Tex., p. 72, 1991.

Livingston P. O. Experimental and clinical studies with active specificimmunotherapy. IN Immunity to Cancer II, M. S. Mitchell (ed), Alan R.Liss, New York, 1989.

Mittelman A., Chen Z. J., Yang H., Wong G. Y., Ferrone S. Human highmolecular weight melanoma-associated antigen (HMW-MAA) mimicry by mouseanti-idiotype monoclonal antibody MK₂ -23: Induction of humoralanti-HMW-MAA immunity and prolongation of survival in patients withStage IV melanoma. Proc Natl Acad Sci USA 89:466-470, 1992.

Morisaki T., Yuzuki D. H., Lin R. T., Foshag L. J., Morton D. L. andHoon D. S. B. Interleukin 4 receptor expression and growth inhibition ofgastric carcinoma cells by interleukin 4. Cancer Res 52:6059-6065, 1992.

Morton D. L., Foshag L. J., Hoon D. S. B., Nizze J. A., Wanek L. A.,Chang C., Davtyan D. G., Gupta R. K., Elashoff R., Irie R. F.Prolongation of survival in metastatic melanoma after active specificimmunotherapy with a new polyvalent melanoma vaccine. Ann Surg16(4):463-482, 1992.

Portoukalian J., Carrel S., Dore J.-F., Rumke P. Humoral immune responsein disease-free advanced melanoma patients after vaccination withmelanoma-associated gangliosides. Int J Cancer 49:893-899, 1991.

Ravindranath M. H., Morton D. L., Irie R. F. An epitope common togangliosides 0-acetylated GD3 and GD3 recognized by antibodies inmelanoma patients after active specific immunotherapy. Cancer Res49:3891-3897, 1989.

Real F. X., Mattes J. M., Houghton A. N., Oettgen H. F., Lloyd K. O.,Old L. J. Class 1 (unique) antigens of human melanoma. Identification ofa 90.000 dalton cell surface glycoprotein by autologous antibody. J ExpMed 160:1219, 1984.

Reisfeld R. A., Cheresh D. A. Human tumor antigens. Adv Immunol0:323-377, 1987.

Rosenberg, S. A., Spiess, P. and Lafreniere, R. A New Approach to theAdoptive Immunotherapy of Cancer with Tumor-Infiltrating Lymphocytes.Science 233:1318, 1986.

Smith L. H., Yin A., Glasky M. S., Tyler N., Robles M., Foster C. A.,Bieber M., Teng N. N. H. Human monoclonal antibody recognizing anantigen associated with ovarian and other adenocarcinomas. Am J ObstetGynecol 166:634-645, 1992.

Szabo A., Dalmau J., Manley G., Rosenfeld M., Wong E., Henson J., PosnerJ. B., Furneaux H. M. HVD, a paraneoplastic encephalomyelitis antigen,contains RNA-binding domains and is homologous to Elav and Sex-lethal.Cell 67:325-333, 1991.

Tai T., Cahan L. D., Paulson J. C., Saxton R. E., Morton D. L., Irie R.F. Enzyme linked immunosorbent assay (ELISA) for the detection of humanantibody to ganglioside GD2. J Natl Cancer Inst 73:627-633, 1984.

Tai T., Paulson J. C., Cahan L. D., Irie R. F. Ganglioside GM2 as ahuman tumor antigen (OFA-1-I). Proc Natl Acad Sci USA 80:5392-5396.1983.

Tai T., Cahan L. D., Tsuchida T., Saxton R. E., Irie R. F., Morton D. L.Immunogenicity of melanoma-associated gangliosides in cancer patients.Int J Cancer 35:607-612, 1985.

Tan E. M. Autoantibodies in pathology and cell biology. Cell 67:841-842,1991.

Vlock D. R, Scalise D., Meglin N., Kirkwood J. M., Ballou B. Isolationand partial characterization of melanoma-associated antigens identifiedby autologous antibody. J Clin Invest 81:1746-1751, 1988.

Watanabe T., Pukel C. S., Takeyama H., Lloyd K. O., Shiku H., Li L. T.C., Travassos L. R., Oettgen H. F., Old L. J. Human melanoma antigen AHis an autoantigenic ganglioside related to GD2. J Exp Med 156:1884-1889,1982.

Woodbury R. G., Brown J. P., Yeh M. H., Hellstrom I., Hellstrom K. E.Identification of a cell surface protein, p97, in human melanomas andcertain other neoplasms. Proc Natl Acad Sci USA 77:2183-2186, 1980.

Yamamoto S., Hoon D. S. B., Chandler P., Schmid I., Irie R. F.Generation of lymphokine-activated killer cell activity by low-doserecombinant interleukin-2 and tumor cells. Cellular Immunol 128:516-527,1990.

Yamamoto S., Yamamoto T., Saxton R. E., Hoon D. S. B., Irie R. F.Anti-idiotype monoclonal antibody carrying the internal image ofganglioside GM3. J Natl Cancer Inst 82:1757-1760, 1990.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 9                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acid residues                                            (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GlnAspLeuThrMetLysTyrGlnIlePhe                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acid residues                                            (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       AspSerArgProGlnAspLeuThrMetLysTyrGlnIlePhe                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Genomic                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       AAAGATCTGATATTTCATAGTCAGATCCTG30                                              (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Genomic                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GACGACGAGCGCGGCGATATCATCATC27                                                 (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acid residues                                            (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       IleMetThrGlnLeuPheGlnAspTyrLys                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Genomic                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GATTCGCGCCCGCAGGATCTGACTATGAAATATCAGATCTTTTAA45                               (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Genomic                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       TTAAAAGATCAGATATTTCATAGTCAGATCCTGCGGCCGCGAATC45                               (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acid residues                                             (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       LysTyrGlnIle                                                                  (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acid residues                                             (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GlnAspLeuThrMetLysTyrGlnIle                                                   15                                                                            __________________________________________________________________________

What is claimed is:
 1. A purified polypeptide segment that includes theamino acid sequence QDLTMKYQI (seq id no:9).
 2. The polypeptide of claim1, further defined as including the amino acid sequence QDLTMKYQIF (seqid no:1).
 3. The polypeptide of claim 1 further defined as comprising amolecular mass of about 43 kDa.
 4. The polypeptide of claim 1 or 2,further defined as having a length of less than 100 amino acids.
 5. Thepolypeptide of claim 4, further defined as having a length of less than50 amino acids.
 6. The polypeptide of claim 5, further defined as havinga length of less than 25 amino acids.
 7. The polypeptide of claim 6,further defined as having a length of 10 amino acids or less.
 8. Thepolypeptide of claim 1, further defined as including the amino acidsequence of β-galactosidase or glutathione-S-transferase.
 9. An antigencomposition comprising an amount of a polypeptide in accordance withclaim 1, effective to elicit a CTL or antibody response.
 10. The antigencomposition of claim 9 wherein the polypeptide is present at aconcentration of between 1 mg/ml and about 10 mg/ml.
 11. The antigencomposition of claim 10 wherein the polypeptide is present at aconcentration of about 5 mg/ml.
 12. The antigen composition of claim 9wherein said polypeptide is a component of a polyvalent melanoma cellvaccine, wherein the cells in said vaccine are rendered inviable.